Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca.

Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.     

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.     

Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation

Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC₅₀) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.     

Antigenic relationships and relative immunogenicities of isolated metalloproteinases from Echis ocellatus venom.

The antigenic relationship between snake venom metalloproteinases (SVMPs) was analysed using rabbit antisera raised against the native forms of two SVMPs purified from Echis ocellatus venom. Using enzyme-linked immunosorbent assay (ELISA), western blotting and two-dimensional SDS-PAGE, our findings show that antibodies raised against EoVMP1, a non-haemorrhagic class P-I 24kDa SVMP, and EoVMP2, a haemorrhagic class P-III 56kDa SVMP, demonstrate cross-reactivities which relate to the domain hierarchy observed in class P-I to P-III/IV SVMPs. A third 65kDa P-III metalloproteinase (designated EoVMP3) was also isolated from E. ocellatus venom using hydrophobic interaction, size exclusion and anion exchange chromatography. In comparative immunoassays, EoVMP2 and EoVMP3 bound strongly to the commercial monovalent ovine Fab fragment antivenom EchiTAbtrade mark (raised against the same venom), but EoVMP1 showed no cross-reactivity. This could indicate that antivenoms may lack antibodies to potentially important venom components.     

Molecular characterization of L-amino acid oxidase from king cobra venom.

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.     

Isolation and characterization of a novel P-II class snake venom metalloproteinase from Trimeresurus stejnegeri.

Stejnitin, a novel class P-II snake venom metalloproteinase (SVMP) with a molecular weight of about 35kDa, was purified from Trimeresurus stejnegeri venom. The cDNA of stejnitin encoded a polypeptide of 295 amino acid residues which comprises a signal peptide, proprotein, metalloproteinase domain, spacer and disintegrin domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIa SVMPs which comprises metalloproteinase and disintegrin together. Results from DNA fragmentation and flow cytometry analysis also indicated that stejnitin is able to induce apoptosis of ECV304 cells (R=0.908, P=0.012).     

Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland.

Hemorrhagic toxins are widely distributed in viperid and crotalid snake venoms. Envenomation of Trimeresurus stejnegeri, a member of Crotalidae family, caused potent systemic and local hemorrhage. Up to now, there is no report on hemorrhage toxins from this venom. In this work, we cloned two cDNAs of P-III metalloproteinase precursors, designated as stejnihagin-A and stejnihagin-B, respectively, from T. stejnegeri venom gland. Both cDNAs encode an opening reading frame of 600 amino acid residues, containing a signal sequence, a proprotein domain, a metalloproteinase domain, a disintegrin-like domain and a cystetine-rich domain. Sequence analysis suggested that these two sequences shared highest similarity to the hemorrhagic toxin HR1b from T. flavoviridis. Aligning the deduced mature protein sequences of stejnihagin-A and stejnihagin-B with other snake venom metalloproteinases (SVMPs), we observed that stejnihagin-A and stejnihagin-B, together with HR1b shared the common cysteinyl residue at the position 100 in the metalloproteinase domain. In combination with the phylogenetic analysis, we presumed that stejnihagin-A, stejnihagin-B and HR1b might constitute a novel subclass of P-III SVMPs, named P-IIIc.     

Immunization with cDNA of a novel P-III type metalloproteinase from the rattlesnake Crotalus durissus durissus elicits antibodies which neutralize 69% of the hemorrhage induced by the whole venom

Zinc-dependent metalloproteinases play a key role in the hemorrhage induced by viperid bite envenoming. In this work we report the cloning and sequencing of the cDNA from a novel P-III type metalloproteinase from Crotalus durissus durissus venom glands. The recombinant plasmid was used for DNA immunization in mice using accelerate DNA-coated microparticles with the Gene Gun system. The results showed that there is no significant difference in the efficiency of the immunization in mice when gold or tungsten microparticles were used. A pool of the sera from mice immunized with the metalloproteinase encoding DNA neutralized the hemorrhagic activity of C. d. durissus venom. The co-immunization with DNA encoding the metalloproteinase and a plasmid encoding the murine IL-2 increased the number of mice which show a specific antibody response towards C. d. durissus venom antigens. The neutralizing ability of the produced antibodies demonstrates that DNA immunization with tungsten microparticles may be used in strategies for antivenom production.     

Molecular cloning and sequence analysis of cDNA encoding flavoridin, a disintegrin from the venom of Trimeresurus flavoviridis.

We isolated a cDNA of 2001bp encoding the full-length precursor of flavoridin, which is one of the four disintegrins in the venom of Trimeresurus flavoviridis, and analyzed the cDNA nucleotide sequence. The deduced amino acid sequence of the open reading frame consisted of a pro-domain (190 residues), a metalloproteinase domain (205 residues), a spacer domain (18 residues) and a disintegrin (flavoridin) domain (70 residues), thus indicating that the flavoridin precursor belongs to the P-II class of snake venom metalloproteinases. The unknown metalloproteinase domain shared strong sequence similarity with HR2a (71.2% identity) and H(2)-proteinase (74.1% identity), a low molecular mass hemorrhagic metalloproteinase and a non-hemorrhagic metalloproteinase in the same snake venom, respectively.     

A snake venom metalloproteinase that inhibited cell proliferation and induced morphological changes of ECV304 cells.

TSV-DM, a basic metalloproteinase with a molecular weight of 110kDa, was purified from Trimeresurus stejnegeri venom. TSV-DM degraded the Aalpha chain of fibrinogen more rapidly than the Bbeta chain in a dose dependent manner. The cDNA of TSV-DM encoded a polypeptide of 622 amino acid residues, which comprises a signal peptide, proprotein, metalloproteinase domain, spacer, disintegrin-like domain and cysteine-rich domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIIb snake venom metalloproteinase, which comprises vascular apoptosis-inducing proteins. TSV-DM inhibited cell proliferation and induced cell morphologic changes transiently of ECV304 cells. However, DNA fragmentation and DNA content analysis demonstrated that this metalloproteinase could not induce ECV304 cells apoptosis.     

Mass spectrophotometric evidence for P-III/P-IV metalloproteinases in the venom of the Boomslang (Dispholidus typus).

The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.     

Acetylcholinesterase from snake venom as a model for its nerve and muscle counterpart.

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. At the neuromuscular junction of vertebrates, for example, it allows a fine temporal control of muscle contraction. The presence of AChE in tissues devoid of cholinergic function is also well known and raises the question of its role. In particular, AChE is abundant in the venoms of Elapid snakes, except mambas. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning of cDNA of the AChE from Bungarus fasciatus venom showed that its C-terminal peptide is very different from those of other AChEs. The partial sequence of the Bungarus fasciatus AChE gene shows that this peptide is encoded by a new alternative exon, called S for soluble and snake. It is a short very basic peptide of 15 residues. Analysis of the venom enzyme and in vitro expression experiments showed that the last eight residues are removed in the mature protein. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to fasciculins from mamba venoms. While Ophiophagus AChE is as sensitive as Torpedo enzyme (IC50 around 10(-10) M), Naja and Heamacatus AChEs are insensitive to the toxin up to a concentration of 10(-6) M Bungarus AChE has an intermediate IC50 of 10(-8) M. Analysis of its sequence reveals two major differences in the peripheral site region, compared to Torpedo or mammalian AChEs: at position 70 it contains a methionine instead of a tyrosine, and at position 285 it contains a lysine instead of an acidic residue (glutamic or aspartic acid). Modification of these residues by site-directed mutagenesis, and enzymatic analysis of modified recombinant enzymes, confirmed that these two residues are implicated in the properties of the Bungarus AChE peripheral site. The presence of an alternative exon, which generates a soluble form of AChE in venoms, raises interesting evolutionary questions: Does it exist in snakes whose venom does not contain AChE, e.g., mambas? Did this exon pre-exist, for expression in other contexts? Snake venom AChEs offer an exceptional system for analyzing the mechanism of peripheral site inhibition, because of their wide range of activities.     

Molecular cloning of HR1a and HR1b, high molecular hemorrhagic factors, from Trimeresurus flavoviridis venom.

HR1a and HR1b are two high molecular weight (P-III class) hemorrhagic factors in the venom of Trimeresurus flavoviridis. In this study, we cloned cDNAs of the HR1a and HR1b precursors and analyzed their nucleotide sequences. The cDNA for HR1a was 2368 nucleotides in length and encoded an open reading frame (ORF) of 609 amino acids; that for HR1b was 2237 nucleotides and encoded an ORF of 614 amino acids. Both cDNAs belonged to the N-III class consisting of signal, pro, metalloproteinase, disintegrin-like and cysteine-rich regions, and shared strong amino acid sequence similarity (74.4%). The HR1b precursor was found to have an additional seven amino acid sequence at the carboxyl terminus compared with a mature form of HR1b.     

Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus

During the characterization of hemorrhagic factor in venom of Rhabdophis tigrinus tigrinus, so-called Yamakagashi in Japan, one of the Colubridae family, a novel metalloproteinase with molecular weight of 38 kDa in the Duvernoy's gland of Yamakagashi was identified by gelatin zymography and by monitoring its proteolytic activity using a fluorescence peptide substrate, MOCAc-PLGLA2pr(Dnp)AR-NH2, which was developed for measuring the well-known matrix metalloproteinase (MMP) activity. After purification by gel filtration HPLC and/or column switch HPLC system consisting of an affinity column, which was immobilized with a synthetic BS-10 peptide (MQKPRCGVPD) originating from propeptide domain of MMP-7 and a reversed-phase column, the N-terminal amino acid sequence of the 38 kDa metalloproteinase was identified as FNTFPGDLK which shared a high homology to Xenopus MMP-9. The 38 kDa metalloproteinase required Zn2+ and Ca2+ ions for its proteolytic activity. In addition, the proteolytic activity was almost completely inhibited by BS-10, a MMP inhibitor, but not by the serine proteinase inhibitors, cysteine proteinase inhibitors and aspartic proteinase inhibitors.Together these results demonstrated that the 38 kDa proteinase is a novel snake verom metalloproteinase (SVMP) containing HExGHxxGxxH motif which possesses high affinity to the BS-10 peptide, into its molecule, and the enzymatic properties are closed to that of MMPs. Based on the results obtained in the present study, we concluded that the 38 kDa metalloproteinase is a novel metalloproteinase whose activity may be regulated by the cysteine switch mechanism, and could be classified as one of the matrix metalloproteinases rather than snake venom metalloproteinases.     

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.     

Simplified procedures for the isolation of HF3, bothropasin, disintegrin-like/cysteine-rich protein and a novel P-I metalloproteinase from Bothrops jararaca venom

HF3 and bothropasin are P-III hemorrhagic snake venom metalloproteinases (SVMPs) of Bothrops jararaca. The DC protein is composed of the disintegrin-like/cysteine-rich domains derived from the autolysis of P-III SVMPs. Here we describe simplified procedures for the isolation of HF3, bothropasin, the DC protein, and BJ-PI, a novel P-I SVMP. The isolated proteins were identified by mass spectrometry. BJ-PI is a potent caseinolytic enzyme devoid of hemorrhagic activity. HF3, bothropasin and BJ-PI show distinct fibrinogenolytic activities.     

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.     

“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase highperformance liquid chromatography using a C₁₈ column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.